JP2001046054A - Alcohol-resistant yeast - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an alcohol-resistant yeast capable of producing the same amount of malic acid or the like as that by a conventional method by selecting the yeast excellently growing when the yeast is succeeded from YNBG agar medium to a malt-extract agar medium, but not growing so good when the yeast is succeeded from YPD agar medium to YNBG agar medium. SOLUTION: This alcohol-resistant yeast excellently grows when the yeast is succeeded from YNBG agar medium (containing 0.67% yeast-nitrogen base, 2% glucose and 2% agar) to a malt-extract agar medium, but does not grow so good when the yeast is succeeded from YPD agar medium (containing 1% malt-extract, 2% peptone, 2% glucose and 2% agar) to the YNBG agar medium. The amounts of formed malic acid and succinic acid in the production of refined rice wine (sake) by using the yeast are same as the ones by using Kyokai no.7 in the same condition. The yeast is deposited as SAM001 strain (FERM P-17373), and liquors are preferably produced by using the yeast strain.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to yeast in the production of alcoholic beverages. In particular, the present invention relates to a yeast in which the production of a large amount of an organic acid such as malic acid, which is a problem in sake production using a conventional alcohol-resistant yeast, has been improved.

[0002]

2. Description of the Related Art Alcohol concentration is 1 in sake mash fermentation.
If it exceeds 0%, some yeasts begin to be damaged by the alcohol generated by themselves, and when the alcohol concentration exceeds 15% and approaches 20%, some of the yeasts are observed to be killed. It is thought that various substances are leaked from the yeast. The leaked intracellular substances impart a taste to the sake produced and increase the quality deterioration rate of the produced sake, so that strains resistant to alcohol have been actively searched for. . For example, Sugano et al. Have isolated wild alcohol-resistant yeast from a brewery (J. Brew. Soc. J.).
apan, 65 , 902 (1970)). Hara et al. Bred and isolated alcohol-resistant yeast in a koji extract medium containing alcohol (J. Soc. Brew. Japan).
an, 71 , 301 (1976)), and from this strain, Association No. 11 distributed by the Japan Brewing Association was born.

[0003] Basic research on leakage of intracellular substances has also been advanced. Goto and Toi paid attention to S-adenosylmethionine (hereinafter abbreviated as SAM), which is not contained in rice and koji but contained in a large amount in the vacuoles of yeast, and detected bacteria of SAM. It has been reported that extracorporeal leakage can be an indicator of yeast intracellular substance leakage in sake mash fermentation (J. Brew. Soc. Japan, 8).
7 , 230 (1992)). In addition, it has been revealed that in the sake mash fermentation using alcohol-resistant yeast, the leakage of SAM is small.

[0004]

At present, the most frequently used alcohol-resistant strain for sake production is the Association 11
And other strains are rarely used. Moreover,
Sake produced by Association No. 11 is Association No. 7 and Association No. 9
No., compared with sake produced by commonly used yeast strains such as the Association No. 701, contains much more malic acid, and has a disadvantage that it has a slightly different flavor as compared to ordinary sake. The Association No. 11 is used for sake production only in limited cases.

Hara et al. Breed and isolate alcohol-tolerant yeast using a cell lysing enzyme, apart from breeding and isolating the above-mentioned alcohol-tolerant yeast. Shows high production of malic acid (J. Brew. Soc. Japan, 7
3 , 408 (1978). Therefore, the emergence of alcohol-tolerant yeast exhibiting new properties for producing sake having a flavor in the category of general sake while retaining the advantage of alcohol-tolerant yeast that the leakage of intracellular substances is small is desired. It is desired to use alcohol-resistant yeasts in yeasts.

[0006]

Means for Solving the Problems In order to solve the above-mentioned problems, alcohol-resistant strains are bred under the condition that high-concentration alcohol is present by continuing to stand for a long time even after completion of sake mash fermentation. Among them, various methods were used to search for alcohol-resistant strains having excellent properties. While trying various searches, among the strains selected on the basis of low SAM leakage in mash fermentation, YNBG agar medium (Difco's yeast nitrogen base 0.67) was selected.
%, Glucose 2%, and agar 2%), it grows well when subcultured on a koji extract agar medium, but it is grown on a YPD agar medium (1% yeast extract, 2% peptone, 2% glucose, 2% agar). It was found that some strains with poor growth existed when the culture was subcultured or subcultured on a YNBG agar medium.
Furthermore, when the sake produced by these strains was examined, it was found that there were many strains producing good sake which had no problem in producing organic acids such as malic acid, and completed the present invention.

[0007] That is, the present invention provides a new type of alcohol-resistant yeast strain that grows poorly when it is subcultured into a YPD medium or YNBG medium after being subcultured into a YNBG medium. Although it is an alcohol-resistant strain, it is possible to produce good sake having an organic acid content such as malic acid equivalent to that of general sake.

[0008] The yeast of the present invention can be obtained by searching for yeast strains separated from those that have been left standing for a long period of time even after the completion of the mash fermentation. here,
The yeast used for mash preparation is Kyokai No. 7, No. 9 or 701.
Various yeasts can be used, including yeast strains belonging to Saccharomyces cerevisiae, which are used in ordinary sake production such as No. 1, and yeasts that have been mutated with a mutagen, ultraviolet rays, radiation, etc. may be used. However, a desired strain can be obtained by a mutation caused by spontaneous induction without performing a mutation treatment.

For the preparation of the mash, rice, koji and water used in ordinary sake production may be used, and if necessary, lactic acid or an enzyme agent may be added. In addition, the method of preparing the mash may be a conventional mash fermentation method as appropriate, and is preferably 1
The fermentation is preferably carried out 8 to 20 days after the distillation at a temperature of 0 to 20 ° C. under fermentation conditions in which the alcohol concentration exceeds 18%. Moreover, it is preferable that the fermentation of the fermented mash is continued at a temperature of 10 to 20 ° C. for 30 days or more. May be placed.

The yeast strain can be separated from the mash which has been kept still for a long period of time even after the fermentation is completed, using a general method. Preferably, the mash is diluted with physiological saline and the koji extract is used. It is preferable to apply the solution to an agar medium, and to incubate the culture at 15 to 30 ° C. statically to form a colony. Then, the colony is preferably transferred to a slant of a koji extract agar medium.

In order to select a strain having a small amount of SAM leakage from the isolated strains, a small preparation of mash fermentation is performed using each of the isolated strains and the parent strain, and the SAM concentration and alcohol concentration of each mash supernatant are changed over time. And the SAM concentration of the mash supernatant when the alcohol concentration reaches 18% is compared with that of the parent strain to determine whether SAM leakage is small. At this time, the SAM concentration is measured, for example, by the method of Goto and Toi (J. Brew. Soc. J.
ap., 87 , 230 (1992)), and the measurement of the alcohol concentration can be performed, for example, according to the method described in the comment of the prescribed analysis method of the NTA.

Next, from among the strains with low SAM leakage obtained above, those strains that do not grow well on the YPD agar medium and the YNBG agar medium are selected from the SAMs described above.
The strain determined to have little leakage was first transferred to a slant of a YNBG agar medium, and then transplanted from the slant to a koji extract agar medium, a YPD agar medium and a YNBG agar medium, and the three kinds of agar medium were heated at 30 ° C. For 3 days, and visually determine the growth state, whereby it is possible to obtain a koji extract agar medium that has good growth but a YPD agar medium and a YNBG agar medium that have poor growth. The koji extract medium mentioned here was prepared from a liquid obtained by adding twice the weight of water to koji rice, allowing the koji rice to stand at 50 ° C. overnight, and saccharifying the rice with the enzyme of the koji mold, followed by filtration. It is a medium. Koji rice is a steamed rice that is allowed to cool to around 40 ° C in a koji room and sprinkled with koji mold to grow the koji mold on the rice, and contains an enzyme that saccharifies rice starch. . In addition, good or bad growth can be judged as good when the bacterial cells increase over the whole inoculated surface, and bad when only small and small lump of bacterial cells are formed on the inoculated surface.

[0013] Thus, when the SAM fermentation is low in the mash fermentation and the YNBG agar medium is subcultured, the YP
A yeast strain having poor growth properties can be obtained on the D agar medium and the YNBG agar medium. The bacterial strain of the present invention thus obtained is a mash supernatant S when the alcohol concentration reaches 18% in normal sake mash fermentation.
The AM concentration was 50% or less of Association No. 7, and the killing rate of yeast by methylene blue staining when the alcohol concentration reached 18% was 10% or less.

The strain of the present invention comprises YPD agar medium and YN
In addition to poor growth on BG agar medium, molasses medium (molasses 1
0%, ammonium sulfate 0.5%, urea 1%, monopotassium dihydrogen phosphate 0.5%, magnesium sulfate 0.05
%), The yield of bacterial cells in aeration and stirring culture was only 20 to 30% as compared with the case of Association No. 7. From these facts, it is considered that the strain of the present invention requires what is contained in the koji extract, although the substance cannot be specifically identified.
No alcohol-tolerant yeast having such auxotrophy has been known so far, and it has been found that the strain of the present invention is a novel type of alcohol-tolerant yeast. Therefore, one of the obtained bacterial strains was named SAM001, and this strain was deposited as Accession No. FERM P-17373 with the Biotechnology Industrial Research Institute, Ministry of International Trade and Industry, Ministry of International Trade and Industry.

The SAM001 strain is a strain obtained by the above-mentioned method using Kyokai No. 7;
-Fertility in alanine medium was the same as that of the parent strain, Association No. 7. In order to examine the strength of the SAM001 strain in alcohol tolerance, 16% alcohol was present in comparison to the parent strain of the present strain, Association No. 7 and the alcohol-resistant strain, Association No. 11, which is most used for sake production. Viability in the presence of, 10% growth in the presence of alcohol and 18%
The fermentation ability of alcoholic mash was measured. As shown in the results in Table 1, it was revealed that the alcohol tolerance ability of the SAM001 strain was different from that of the Association No. 11.

[0016]

[Table 1]

The present invention also includes alcoholic beverages produced using the yeast of the present invention. The yeast of the present invention can be used for the production of various alcoholic beverages such as sake, shochu, beer, wine, and the like, and the production of alcoholic beverages using the bacterial strain of the present invention is performed by appropriately employing the conventional alcoholic beverage production method. It is possible by using.

Among the alcohol-tolerant strains, sake produced by Association No. 11 which is most used for sake production is sake produced by ordinary sake yeast such as Association No. 7, Association No. 9 and Association No. 701. It is known that malic acid and the like are contained in a large amount and the organic acid composition is different as compared with the above.However, when an organic acid analysis was performed on sake produced by the strain of the present invention, various acids including malic acid were There were many strains whose organic acid content was equivalent to that of the parent strain (Kyoto 7).

The quality of the produced sake was determined by sensory evaluation.
There was more sake taste than the one made by No. In addition, when good flavor was determined by sensory evaluation, there were many that were superior to those of the parent strain. From this, it was presumed that a large number of bacterial strains having the flavor of conventional sake and capable of producing sake with good sake quality were included.
Hereinafter, the present invention will be described with reference to specific examples, but the present invention is not limited to the following specific examples.

[0020]

Example 1 Yeast No. 7 was inoculated into 10 ml YPD medium and cultured at 30 ° C. for 1 day. This culture was added to 400
The cells were transplanted into a ml molasses medium and cultured at 30 ° C. for 3 days.
The culture solution was collected and washed, and the sake mash fermentation was performed using the yeast cells and the blended ingredients shown in Table 2.

[0021]

[Table 2]

The rice used as the raw material was Nipponbare (produced in 1995), which was polished to a polishing rate of 70%. In addition, koji was made in a koji room for 2 days by a conventional method, and spitase MK was used as an enzyme preparation. The preparation method was divided into 3 days, the first day was the first soup, the first day was left, the third day was the middle soup and the fourth day was the soup.
Fermented at 0 ° C. At the time of the initial addition, 1 g of yeast was added by wet cell weight.

On the 17th day after the distillation, the alcohol concentration of the mash supernatant was 1
Reached 9.3%. Thereafter, the mixture was allowed to stand still at 15 ° C., the moromi was sampled 60 days after the distillation, and a liquid diluted with sterile physiological saline was applied to the koji extract agar medium. Add this agar medium to 3
The culture was allowed to stand still at 0 ° C. for 4 days to form colonies, and a slant of a candidate strain for searching for an alcohol-resistant strain was prepared from each colony.

Using the yeast SAM001, which was cultured by the above method using the SAM001 strain of the present invention, which is one of the obtained strains, and the parent strain, Association No. 7, was collected and washed. Sake mash fermentation was carried out with the blending composition shown in Table 2. At this time, the alcohol concentration and the SAM concentration of the mash supernatant were measured, and the results are shown in Tables 3 and 4, respectively.

[0025]

[Table 3]

[0026]

[Table 4]

As shown in Table 3, the alcohol production rate of the strain of the present invention was slightly slower in the first half of the mash fermentation than in the parent strain, but was faster in the second half of the mash fermentation than in the parent strain. Overtake production. Further, as shown in Table 4, the amount of SAM leakage of the strain of the present invention was significantly smaller than that of the parent strain, and the alcohol concentration was 18%.
When the amount of SAM leaked was compared based on the SAM concentration at the time when the strain reached, the amount of SAM leaked from the strain of the present invention was 3% of that of the parent strain.
1.6%.

Here, the SAM concentration was measured by a method using high performance liquid chromatography of Goto and Toi (J. Bre
w. Soc. Japan, 87 , 230 (1992))
It went according to. That is, a strong cation exchange resin column Chemopakak Nucleosil 10SA
And developed with a gradient of 0.05 M to 0.5 M of ammonium phosphate buffer (pH 3.0) using 26
The detection was performed by detecting the absorbance at 0 nm.

[0029]

Example 2 The strain SAM001 of the present invention and the parent strain Kyokai No. 7 were inoculated on a YNBG agar medium and allowed to stand at 30 ° C. for 4 days to produce a slant. The slant was transplanted to a koji extract agar medium, a YPD agar medium, and a YNBG agar medium, cultured at 30 ° C. statically, and three days later, the growth status was visually determined. Table 5 shows the results.

[0030]

[Table 5]

As shown in Table 5, the parent strain, Association No. 7, grew well in all media. Although the strain of the present invention grew well on the koji extract agar medium, the thickness of the cell mass formed on the agar surface was higher on the YPD agar medium and the YNBG agar medium than on the parent strain, Association No. 7. Was thin and the growth was poor. In particular, in the growth of the YNBG agar medium, the cells of the parent strain grow on the entire surface of the portion inoculated with the bacteria, whereas the cells of the strain of the present invention show a small number of small cells. It was observed that there were only lumps and clearly poor growth.

[0032]

Example 3 The strain, parent strain and Kyokai No. 11 of the present invention were inoculated in a koji extract medium and cultured at 30 ° C. for 1 day. Alcohol was added to these cultures so that the alcohol concentration became 16%, incubated at 30 ° C. for 4 hours, and the mortality was measured by methylene blue staining. Table 6 shows the results.

[0033]

[Table 6]

As shown in Table 6, the ability of the strain of the present invention to maintain viability at a high alcohol concentration was not as strong as Association No. 11, but was stronger than the parent strain.

[0035]

EXAMPLE 4 The strain, parent strain and No. 11 of the present invention were inoculated in a koji extract medium and cultured at 30 ° C. for 1 day. These cultures were transplanted to a 10% alcohol-containing koji extract medium at a yeast density of about 10 ⁴ / ml.
The culture was allowed to stand still for 0 hour, and the number of viable cells was measured using a toma blood cell plate to determine the growth rate. Table 7 shows the results.

[0036]

[Table 7]

As shown in Table 7, the growth rate of the strain of the present invention at a high alcohol concentration was faster than that of the parent strain and Kyokai No.11.

[0038]

Example 5 A strain of the present invention was added to 10 ml of a koji extract medium in 4 ml.
The inoculum was cultured at 30 ° C. for 1 day. This culture solution 1
This was transplanted to a koji extract medium (500 ml) and cultured at 30 ° C. for 2 days to prepare a bacterial solution of the strain of the present invention grown in a total of 2 L of medium. After adding this bacterial solution to a container containing 100 L of water and 1 L of lactic acid, 45 kg of koji rice and then 105 kg of 70% polished rice were added, and a quick-brewed sake mother was prepared by a conventional method. Using this prepared sake mother, brewing was carried out with the blended composition shown in Table 8.

[0039]

[Table 8]

Here, rice as a raw material is Nipponbare (Heisei 10
The koji was produced in a koji room for 2 days by a conventional method. The preparation method was divided into 3 days, the first day was the first addition, the first day was left, the middle day was added on the third day, the fourth day was added, and the fermentation was carried out at 15 ° C. The sake mother added the whole amount prepared at the time of the first addition.

On the 19th day, 30% alcohol was added to 87%.
Add 6 L, pump 120 kg of rice on the 20th and pump 240 L of water
Among them, saccharification was performed at 55 ° C for 12 hours using an enzyme agent spitase M, and the mixture was cooled to 15 ° C and added in four stages. On the 21st day of the distillery, the upper tank was made, and the alcohol concentration was 20.7%, sake degree +7.5, acid degree 1.7, and amino acid degree 1.7.
L was obtained.

The sake brewed in the same manner using the parent strain had an alcohol concentration of 20.4%, a sake degree of +5.0,
The acidity was 1.8 and the amino acid content was 1.5. Compared with the sake degree, sake produced from the strain of the present invention was high, and it was revealed that dry type sake could be produced using the strain of the present invention. .

The glucose and maltose concentrations of the present sake were 1.82% and 0.30%, respectively. Compared to 2.26% and 0.45% of sake brewed by the same method using the parent strain, the sake produced by the strain of the present invention has less residual glucose and maltose, and produces dry-type sake. It is considered suitable for

When the kill rate of yeast in the mash immediately before the upper tank was observed by methylene blue staining, 19.2%
5 when brewed by the same method using the parent strain.
It was much lower than 8.3%. The kill rate when the alcohol concentration reached 18% was 1.4%, which was 15.0 in the case of brewing in the same manner using the parent strain.
It was considerably lower than the percentage.

The SAM concentration of the mash supernatant when the alcohol concentration reached 18% was 4.6 ppm, which was considerably lower than 12.6 ppm when brewed by the same method using the parent strain. Honsei Sake, Parent Stock and Association 11
The organic acid analysis of the sake brewed in the same manner as in the present brewed sake using No. No. 10 gave the results shown in Table 9.

[0046]

[Table 9]

From the results shown in Table 9, the sake produced by the Association No. 11 contains a much larger amount of malic acid and succinic acid than that of the Association No. 7, but it is produced by the strain of the present invention. It was confirmed that the organic acid composition of the sake did not differ much from that of the parent strain, Association No. 7.

[0048]

Example 6 Using the strain of the present invention described in Example 5, sake produced by Kyokai No. 7 and Kyokai No. 11, a panel of 15 panelists was subjected to a sensory evaluation to determine whether or not a panel of 15 persons had a flavor like sake. . The evaluation method is based on the sake produced by the Association No.7. 5: The sake is much more refined than the sake produced by the Association No.7. 4: The sake is slightly more refined than the sake produced by the Association No.7. Probable 3: Equivalent to sake made by Association No. 2: Slightly less like sake compared to sake made by Association No. 7, 1: Completely compared to sake made by Association No. 7 Performed on a five-point scale, not like sake.
The results shown in Table 10 were obtained.

[0049]

[Table 10]

As shown in Table 10, the sake produced from the strain of the present invention had a flavor more like sake than the sake produced by Association No. 11.

[0051]

Example 7 Using the strain of the present invention described in Example 5, the sake produced by Kyokai No. 7 and Kyokai No. 11,
A sensory evaluation was performed to determine whether the name panel had a good quality flavor. The evaluation method is 5: very good, 4: somewhat good, 3:
Normally, the evaluation was performed on a five-point scale of 2: somewhat poor, 1: very poor, and the results shown in Table 11 were obtained.

[0052]

[Table 11]

As shown in Table 11, the sake produced from the strain of the present invention has a slightly better flavor than the sake produced by the Association No. 7, and has a higher quality than the sake produced by the Association No. 11. Had a flavor.

Claims (4)

[Claims] Claims 1. Although growth is good when subcultured from YNBG agar medium to koji extract agar medium, growth is poor even when subcultured from YNBG agar medium to YPD agar medium or YNBG agar medium. Alcohol-resistant yeast. 2. The production amount of malic acid and succinic acid in sake production is equal to that in a case where sake production is performed under the same conditions using Association No. 7 under the same conditions.
The yeast according to 1. 3. The yeast according to claim 2, which is SAM001.
Strain (FERM P-17373). 4. Alcoholic beverages produced using the yeast strains according to claim 1, 2 and 3.